Why is exercise such an effective therapy for conditions like obesity, type II diabetes and sarcopenia? The pathways responsible for the benefits of exercise represent therapeutic targets with enormous translational potential for impacting the health of billions of people. However, the identities of these pathways remain to be fully elucidated. A powerful new paradigm has emerged for explaining exercise's benefits based on a poorly defined endocrine function of skeletal muscle. Skeletal muscles can secrete peptides called myokines (e.g., IL- 6, myostatin, irisin), that work in a beneficial hormone-like fashion locally on skeletal muscle itself and on neighboring and distant organs. Myokines represent potential therapies for many conditions such as diabesity, sarcopenia and muscular dystrophy. Many myokines are secreted via a Golgi complex-mediated pathway; however the mechanisms governing myokine secretion at the Golgi remain unknown. We hypothesize that Golgi nNOSb is a novel regulator of myokine secretion. Our hypothesis is supported by our published and preliminary data showing that nNOSb is targeted to Golgi in skeletal muscle and that nNOS regulates expression of several myokines. Our hypothesis is also supported by human studies suggesting nitric oxide is necessary for exercise-driven IL-6 expression and secretion. To test our hypothesis we are generating skeletal muscle-targeted nNOSb knockout (KO) mice with startup funds anticipated to be ready before proposal activation. First, we propose to validate targeted inactivation of skeletal muscle nNOSb and determine its phenotypic impact (Aim 1). This is necessary to ensure model accuracy and because skeletal muscle nNOSb functions are unknown, although indirect evidence suggests nNOSb regulates muscle growth and exercise performance. Then, to test our hypothesis that Golgi nNOSb regulates myokine secretion, we will use a biased candidate-based approach to evaluate secretion of known myokines into the circulation of nNOSb KO mice at rest and subjected to acute running exercise. Many myokines are yet to be identified in vivo; therefore we will also use an unbiased mass spectrometry-proteomics strategy to identify nNOSb-regulated myokines secreted from resting and active muscles. In this way we will identify Golgi nNOSb-regulated myokines secreted at rest or during exercise (Aim 2). Successful completion of these aims will generate novel insights into nNOSb function and into the pathways regulating muscle growth and exercise performance. Such outcomes would support nNOSb's consideration as a new therapeutic target for conditions such as muscular dystrophy. We also expect to uncover a novel nNOSb-dependent mechanism governing skeletal muscle myokine secretion. By determining a role for nNOSb in myokine secretion, we will gain new insights into skeletal muscle's endocrine function, an exciting new frontier in skeletal muscle biology, that have broader implications for understanding the systemic benefits of exercise. These findings will be used as preliminary data and to develop new hypotheses about nNOSb function for the PI's first R01.